Title

Author

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Torchia, Joseph

Abstract

Cytosine methylation (5mC) is essential for transcriptional control and genomic stability and is often used as a prognostic marker in cancer. Although 5mC has long been considered a relatively stable epigenetic mark, recent studies have demonstrated that it can be reversed enzymatically by TET proteins which oxidize 5mC into 5-hydroxymethylcytosine (5-hmC), and then to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5caC). This mechanism is known as active DNA demethylation and the base excision repair enzyme Thymine DNA Glycosylase (TDG) plays an essential role in this process by removing 5-fC and 5-caC which are subsequently replaced by the unmethylated cytosine. Importantly, homozygous loss of TDG in mice causes embryonic lethality and the observed defects are consistent with a central role for TDG in active demethylation and protein scaffolding events. I hypothesize that the dual catalytic and protein scaffolding activities of TDG are essential for gene transcription and that TDG functions as a tumor suppressor in vivo by maintaining epigenetic stability in cells.

In this study, I investigated the regulation of Hypermethylated in Cancer 1 (Hic1) tumor suppressor to demonstrate that the dual catalytic and scaffolding activities of TDG are essential for gene transcription of HIC1. In addition, I developed a conditional TDG knockout mouse model to determine the role of TDG in development. Hic1 transcription involves a transient accumulation of 5fC/5caC metabolites and the recruitment of an RAR/RXR complex, both of which were found to be TDG dependent. Tdg deletion in vivo causes Hic1 silencing via promoter DNA hypermethylation. In addition, a loss of TDG in adult mice predisposes them to a high prevalence of hepatocellular carcinoma (HCC), independent of cirrhosis or fibrosis. Surprisingly, HCC occurred predominantly in male mice, which also showed increased obesity compared to age matched controls. RNAseq analysis of livers from TDG deleted mice showed downregulation of several metabolism relevant genes. These findings are consistent with an onset of HCC via the non-alcoholic fatty liver pathway. Collectively, this study shows that the dual catalytic and scaffolding activities of TDG are required in gene transcription events, and provides the first evidence of TDG as a tumor suppressor of liver cancers.